1. Field of the Invention
This invention pertains to novel porphycene compounds having effectiveness in the treatment and eradication of microbial populations, and other anti-microbial properties, as well as compositions comprising the same, and methods of using these porphycenes to treat microbial infection.
2. Discussion of the Background
During the past three decades an accelerating volume of research has been conducted and published concerning the use of photodynamic therapy (PDT) in treatment of hyperproliferative disease. Only one product had received U.S. regulatory approval prior to December, 1998, and this, a first generation dye mixture of uncertain composition, possesses acknowledged deficiencies in clinical applications for limited approved uses in treatment of various cancers. Since 1970, numerous photoactivatable compounds have been synthesized and studied in vitro and in vivo in the search for superior, second generation dyes. Much of this work has centered upon the porphyrinoid dyes containing a tetrapyrrolic macrocycle as the light energy absorbing moiety. One unique ring system is exemplified by the porphycenes, also cyclic tetrapyrroles, though isomeric with the naturally occurring porphyrin ring system. A selective summary of relevant patent and scientific literature references to PDT research and clinical applications is covered, for example, in U.S. Pat. No. 5,610,175, Vogel et. al., where each references cited in turn offers many more references.
Phase II or Phase III clinical studies are being conducted with a variety of photosensitisers or precursors including a porphycene (Cytopharm), an etiopurpurin (Miravant Technologies), a benzoporphyrin (QLT Phototherapeutics), .delta.-aminolevulinic acid (DUSA), chlorins (Nippon Petrochemical and Scotia), and an expanded porphyrin system (Pharmacyclics), among others. .delta.-Aminolevulinic acid (ALA) is not a photoactivatable dye itself but undergoes endogenous in vivo metabolism to the active molecule protoporphyrin IX (Pp=IX). All of these photosensitizing dyes under clinical development, except the porphycene and ALA, appear to be administered systemically, based on the published literature. Thus, of the tetrapyrroles, only the porphycenes appear to lend themselves to topical application, which is a favored route of administration for several medical treatments, as exemplified by dermatological diseases.
For PDT treatment of microbial infections, as in periodontal disease, and for wound sterilization in hospital or field conditions, human or animal, a desirable photoactivatable agent should possess preferably the following properties:
Application as a topical formulation PA1 Ability to penetrate or bind to bacterial cell walls PA1 Non-toxic to host cells PA1 Rapid metabolism if escape to vascular system occurs PA1 Efficacy against fungal organisms and yeasts PA1 Efficacy against both Gram positive and Gram negative bacteria PA1 Efficacy in destruction of antibiotic-resistant bacteria PA1 Excellent kill of a wide range of microorganisms PA1 Fast-acting, allowing light radiation within minutes of application PA1 Efficacy at low concentration of dye PA1 Efficacy with lower light dose than will destroy host cells
ALA requires several hours to undergo metabolism to Pp-IX and is therefore unsuitable for PDT when only a brief period can elapse between dye application and radiation with light.
The early porphycenes synthesized by Vogel et al. (Angew. Chein. Int. Ed. Eng., 26, 928-931 (1987)) carried an alkyl substituent on each of the four pyrrole rings. In vivo assays revealed that these lipophilic molecules generally needed 12-48 hours to attain the optimal concentration in tumor tissue with highest selectivity for tumor versus non-tumor tissue (Guardiano et al. Cancer Letters, 44, 1-6 (1989)). Conversely, porphycenes synthesized later bearing an alkoxyalkyl substituent on each pyrrole ring were shown to penetrate hyperproliferative cells extremely rapidly, allowing tumor destruction by PDT procedures with light radiation just minutes after dye administration. (Richert et al., J. Med. Chem, 37, 2797 (1994) and Richert et al., Photodynamic Therapy and Biomedical Lasers, Excerpta Medica, 1011, 702, (1992)). Subsequently, independent work confirmed this attractive property conferred by alkoxyalkyl substituents in other porphyrin molecules (Bellnier et al., J. Photocheni. Photobiol. B Biol, 20, 55-61 (1993)). Thus, a class of porphycenes is available which offers both fast-action and permits topical application.
For anti-microbial activity the selected photoactivatable agent needs to penetrate or bind to the bacterial cell wall. There is work by Malik et al,. (J. Photochem. Photobiol., B. Biol., 5, 281-293 (1990)), by Bertoloni et al.,(FEMS Microbiological Letters, 7, 149-156 (1990)) and by Nitzam et al.,(Photochem. Photobiol., 55, 89-96 (1992)) showing that neutral or anionic porphyrinoids are unable to induce the photinactivation of Gram (-) bacteria unless some agent permeabilizing their outer wall is previously added. Malik et al also showed that a cationic peptide (polymixin B) destabilizes the outer wall by replacing Ca.sup.2+ ions so that the permeability of the outer wall is increased and the photosensitizer can penetrate to reach the cytoplasmic membrane. Hitherto no cationic porphyrinoid photosensitizers have been synthesized which meet all the criteria listed above to qualify as suitable photoactivatable dyes for PDT of microbial infections.
Attempts to inactivate Gram negative bacteria by exposure to light plus other porphyrinoid photosensitizers, including Photofrin.RTM., verteporfin (BPD), temporfin (m-THPC), phthalocyanines, chlorins, etiopurpurins, etc. have proven unsuccessful.
In spite of the progress made in the area of photactivated anti-microbial agents, improvements in such agents are sought.